Enzymic synthesis of O-β-d-glucopyranosyl-(1→6)-d-galactose

1. The enzymic synthesis of O-β-d-glucopyranosyl-(1→6)-d-galactose has been described and evidence for the structure presented. 2. It has been shown that the transglycosylase of A. niger provides a convenient means of synthesizing (1→6)-linked disaccharides.     

Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.     

Conformation of (1→3)-α-D-Glucan Tribenzoate

The molecular conformation of (1→3)-α-D-glucan tribenzoate (TBG) was studied by X-ray diffraction measurements coupled with a conformational analysis. Although the fiber pattern obtained was of very low crystallinity, the presence of a meridional reflection at the 5th layer line indicated that the TBG molecule took a five-fold helical conformation with a 19.63 A fiber repeat. A conformational analysis on the five-fold helix, which was done by calculating van der Waals’ repulsion energy between non-bonded atoms comprising the TBG chain, suggested that the most preferable energy-based conformation was –5/1, a left-handed five-fold helix.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

Probe design and synthesis of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of N-glycan

Synthesis and clusterization of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of the N-glycan, as the molecular probes for their biological evaluation, are reported. Key step is the quantitative and the completely α-selective sialylation of the C5-azide N-phenyltrifluoroacetimidate with the disaccharide acceptor, Galβ(1→3)GlcNTroc. Clusterization of the 16 molecules of trisaccharide motif was also achieved by the ‘self-activating click reaction’. These probes could efficiently be labeled by biotin and/or other fluorescence- or radioactive reporter groups through either cross metathesis, acylation, Cu(I)-mediated Huisgen [2+3]-cycloaddition, or the azaelectrocyclization to utilize the various biological techniques.     

Conformational properties of a cyclic oligosaccharide: cyclotrikis-(1→6)-[α-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl]

The title compound is a cyclic oligosaccharide having six glucopyranose residues linked alternatively by -(14) and -(16) glycosidic linkages. Like cyclodextrin analogues it is expected to exhibit an internal cavity and to form inclusion complexes with other species. In order to investigate its conformational preferences, an extensive conformational search was carried out using a combination of Metropolis Monte-Carlo (MMC) procedure in the glycosidic torsion angle space and molecular mechanics procedures. To this end a specific program (METROCYCLIX) was developed. To reduce the MMC search, conformational maps of parent disaccharides were considered as starting entries. Fully minimized conformations were gathered into families using a clustering technique based on RMS fitting over the glycosidic torsion angle values. A wide range of local energy minima were identified in spite of ring closure conditions that constrained the structure of the oligosaccharide. Low energy conformers were stabilized by intramolecular interactions between distant residues. From the Bolzmann population of the best structures derived from the clustering results, various average properties were calculated and compared with experimental data obtained by high resolution NMR. Interpretation of these experimental values (heteronuclear coupling constants, rotating frame nuclear Overhauser effects, relaxation times) relies on the use of Karplus like equations (coupling constants) and analysis of the full relaxation rate matrix treatment (ROE). The quality of the molecular modelling strategy used is assessed by the agreement obtained between calculated and measured observables.     

Molecular and crystal structure of the regenerated form of (1→3)-α-d-glucan

The crystal structure of a regenerated form of (1→3)-α-d-glucan, obtained by solid state deacetylation of the triacetate derivative, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. The structure crystallizes in an orthorhombic unit cell with parameters $\text{a = 16.46}\text{A}\text{?}\text{, b = 9.55}\text{A}\text{?}$ and c (fibre repeat)=8.44 Å, and space group P2₁2₁2₁. The chain conformation is nearly completely extended and is very close to a 2/1 helix, even though the dimer residue is the crystallographic repeat unit. An intramolecular O(2)  O(4)′ hydrogen bond stabilizes the conformation and extensive intermolecular hydrogen-bonding abilizes the packing. The resulting structure is sheet-like, with an alternating polarity of chain directions within the sheet. In its sheet-like character, extensive hydrogen-bonding, and insolubility in water, this polymorph of (1→3)-α-d-glucan resembles regenerated cellulose. The reliability of the structure analysis is indicated by the X-ray residual R=0.206.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A_1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K_m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d-glucose into the growing α-d-glucan and the synthesis of high molecular-weight polymers. However, the α-d-glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than IIa.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Synthesis of methyl α-d-glucopyranosyl-(1–2)-α-d-galactopyranosyl-(1-3)-α-d-glucopyranoside and an acyclic analogue thereof for probing the carbohydrate-binding specificity of bacteriophage ϕX 174

The title trisaccharide was synthesized using methyl 1-thioglycoside building blocks. An acyclic analogue, methyl 3-O-(α-D-glycopyranosyl-oxyethyl)-α-D-glucopyranoside, which has an ethylene bridge in place of the galactosyl residue, was also synthesized.     

MMC and LD simulations of α-D-Glcp-(1→2)-α-D-Glcp-(1→3)-α-D-Glcp-OMe. A model for the terminal trisaccharide in glycoprotein precursors

The conformational flexibility and the dynamics of -D-Glcp-(12)--D-Glcp-(13)--D-Glcp-OMe (I) has been investigated by Metropolis-Monte Carlo with the HSEA (Hard Sphere Exo-Anomeric) force field and Langevin dynamics simulations employing two different CHARMm (Chemistry at HARvard Molecular Mechanics) force fields, CHEAT95 and PARM22. The conformational space spanned by the molecule is similar for the two former force fields but differ significantly for the latter. Hydrogen bonding between O2 and O4 of the title compound is analysed in comparison to NMR and preliminary results from X-ray powder diffraction studies. © 1998 Rapid Science Ltd     

Synthesis of Maltosyl(α1→6)cyclodextrins through the Reverse Reaction of Thermostable Bacillus acidopullulyticus Pullulanase

Maltosyl(α1→6)α-, β or γ-cyclodextrin was synthesized from maltose and α-, β- or γ- cyclodextrin, respectively, using Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). More than 40% of each cyclodextrin substrate was converted to the corresponding maltosyl(α1→6)cyclodextrin under the conditions given below; the combined concentration of maltose and cyclodextrin was 70 ~ 75 % (w/w), the molar ratio of maltose to cyclodextrin was 9~18, and the amount of pullulanase was 100~200units/g of cyclodextrin. The optimum pH and temperature for the formation of maltosyl(α1→6)cyclodextrins were 4.0—4.5 and 60~70°C, respectively. Each maltosyl(α1→6)-cyclodextrin produced was separated from noncyclic saccharides, maltose and branched tetraose, by methanol and ethanol precipitations. The maltosyl(α1→6)cyclodextrins were further purified by gel filtration on a Toyopearl HW 40 S column and crystallization from aqueous (for maltosyl(α1→6)β-cyclodextrin) or methanol (for maltosyl(α1→6)β-cyclodextrin) solution. From 10 g each of the corresponding cyclodextrin, the yields of the purified maltosyl(α1→6)α-, β- and γ-cylcodextrins were 3.0 ~ 3.6 g, 2.5 ~ 2.8g and 2.2 ~ 2.5 g, respectively. Identification of the maltosyl(α1-6)cyclo-dextrins was performed by means of hydrolysis with Klebsiella pneumoniae pullulanase, methyla- tion analysis and ¹³C-NMR analysis.     

The synthesis of O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→4)-N-acetylnormuramoyl-l-α-aminobutanoyl-d-isoglutamine

Benzyl 2-acetamido-3-O-allyl-6-O-benzyl-2-deoxy-4-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)- α-d-glucopyranoside (4) was obtained in high yield on using the silver triflate method in the absence of base. Compound 4 was converted in six steps into benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucopyranosyl)-6-O-benzyl-3-O-(carboxymethyl)-2-deoxy-α-d- glucopyranoside, which was coupled with the benzyl ester of l-α-aminobutanoyl-d-isoglutamine and the product hydrogenolyzed to afford the title compound. O-Benzylation of benzyl 2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-3-O-allyl-6-O-benzyl-2-deoxy-α-d-glucopyranoside with benzyl bromide and barium hydroxide in N,N-dimethylformamide is strongly exhanced by sonication of the reaction mixture.